Influenza viruses rapidly accumulate mutations in antibody (Ab) binding sites within the hemagglutinin (HA) and neuraminidase (NA) proteins, a process termed 'antigenic drift'. Due to antigenic drift, humans are frequently re-infected with antigenically distinct influenza strains and vaccines must be updated frequently. Influenza vaccines fail to elicit protective Ab responses in some individuals, even when vaccine strains are well matched to predominant circulating strains. Elderly individuals, who typically have extensive influenza exposure histories, respond particularly poorly to influenza vaccines. As early as the 1950's, it was noted that the human immune system preferentially mounts Ab responses to previously circulating influenza strains, as opposed to new Ab responses that exclusively target newer viral strains. This process, termed 'original antigenic sin (OAS)', has been proposed to contribute to vaccine failures in individuals with extensive influenza pre-exposure histories. We hypothesize that strain-specific Abs that recognize variable epitopes located on the top of HA are more efficient at neutralizing virus compared to OAS-induced Abs that recognize epitopes in conserved regions of HA. Previous studies of OAS have utilized influenza strains that have dramatic antigenic differences, however humans are more typically sequentially infected or vaccinated with influenza strains that have subtle antigenic changes compared to previously circulating strains. In this proposal, we will determine if OAS is induced by sequential infections with seasonal influenza strains with moderate antigenic differences. We will sequentially infect mice and ferrets with A/Puerto Rico/8/34 viruses with well defined HA antigenic mutations, and we will identify OAS-induced Abs through hemagglutination-inhibition (HAI), ELISA, ELISPOT, FACS-based Ab binding, and in vitro neutralization assays. We will complete similar vaccination experiments in pre-exposed animals with and without the MF59 adjuvant. We will then map the precise binding footprints of anti-HA mAbs derived from mice sequentially infected with distinct influenza strains. We will also map the binding footprints of a large panel of H3N2 mAbs derived from vaccinated humans with different H3N2 pre- exposure histories. Finally, we will determine in vivo neutralization efficiencies of OAS-induced murine and human mAbs using a mouse model. Collectively, these studies will determine (1) if seasonal vaccines induce OAS, (2) if OAS contributes to decreased vaccine efficacy in pre-exposed populations, and (3) if OAS Ab repertoires can be altered by MF59 adjuvants.